N,N’-Dinitrosopiperazine-mediated Ezrin Phosphorylation Involves In Invasion And Metastasis Of Nasopharyngeal Carcinoma6-10B Cells

Objective:To determine the molecular mechanism of N,N’-Dinitrosopiperazine (DNP) increasing the invasion and metastasis of nasopharyngeal carcinoma (NPC)6-10B cells, and identify whether DNP induce the motility and invasion behavious through increasing Ezrin or phos-Ezrin at Threonine567expression.Methods:1. NPC cell lines,6-10B with low metastasis,5-8F with high metastasis, which derived from cell line SUNE-1, were served as cells model. The untreated6-1OB cells and5-8F cells served as control groups.2. Different concentrations DNP were prepared by serial dilution.6-10B cells were treated with different concentrations of DNP. To determine the non-cytotoxic concentration (NCC) of DNP, the Methylthiazolyltertrazolium (MTT) assay was performed to determine the cell viability after DNP treatment. To further evaluate the non-cytotoxic concentration of DNP in NPC cells, LDH activity in cell culture media was detected through the automatic biochemical analyzer after DNP treatment.3.6-10B cells were treated by DNP at non-cytotoxic concentration. To clear that DNP induced Ezrin or phos-Ezrin at Threonine567expression. Western-Blotting was use to detect the expression of Ezrin and phos-Ezrin at Threonine567in6-10B cells after DNP treatment.4. To clarify whether DNP induce NPC cell metastasis, the invasion and migration of6-10B cells were observed by scratch assay and transwell invasion and migration assay after DNP treatment at non-cytotoxic concentration.5.6-10B cell metastasis in BALB/c nude mice was constructed through tail vein, and then treated with DNP by subcutaneous injection of DNP. After DNP treatment, the nude mice were anatomised to observe the metastasis of the6-10B cells. The metastasis of6-10B cells was confirmed through routine pathologic examination. DNP-mediated metastasis of nasopharyngeal carcinoma was analyzed in vivo.6. Ezrin and phos-Ezrin at Threonine567expression in metastatic carcinoma tissues of nude mice were detected by immunohistochemistry SP method, and determined whether DNP promote NPC metastasis through mediating expression of Ezrin and phos-Ezrin at Threonine567in vivo.7. SPSS17.0was used to analyze the data.Results:1. MTT assay and LDH test results showed that DNP had no significant effect on the proliferation of6-10B cells at the concentrations of0～100μmol/L(P<0.05), and so0～100μmol/L is the non-cytotoxic concentration of DNP to NPC cell6-10B.2. Western-Blotting results showed that total Ezrin expression did not alter in6-10B cell after DNP treatment at different concentrations of DNP (P>0.05); while phos-Ezrin at Threonine567expression levels were significantly higher in6-10B cells with DNP treatment than non treatment (P<0.05). Interestedly, phos-Ezrin at Threonine567gradually increased with dose increased and processing time prolonged, showing a significant dose and time dependence.3. The scratch test results showed that the migrating cells with DNP treatment were significantly more than untreated group. Transwell migration and invasion experiment data showed, after being treated with DNP,6-10B cells which passed through the filter membrane were significantly more than the untreated group (P<0.05), which indicated that DNP could enhance NPC6-10B cell migration and invasion ability.4.6-10B cell metastasis in BALB/c nude mice showed that6-10B cell metastasis dramatically increased after treated with DNP, and metastatic tumors were observed in the nude mice lung tissue. And then these NPC cell metastases to lung were confirmed by routine pathological examination, which suggested that DNP could promote6-10B cells lung metastasis.5. The Immunochemistry data showed that phos-Ezrin at Threonine567expression was significantly higher in metastatic carcinoma tissue treated with DNP than that untreated groups (P<0.05), while total Ezrin expression did not alter. These indicated that DNP could promote6-10B cells metastasis by increasing phos-Ezrin at Threonine567expression.Conclusions:1. Phos-Ezrin at Threonine567levels were significantly increased in NPC6-10B cells after DNP treatment, and these expression increased at dose and time manner, but had no effect on the total ezrin expression. This suggests that DNP induced the expression of phos-Ezrin at Threonine567(phos-Ezrin Thr567).2. The migration, motility and invasion ability of6-10B cells were significantly enhanced after treated with DNP. These indicated that DNP promotes nasopharyngeal carcinoma cell invasion and metastasis ability.3. We successfully established metastasis model of6-10B cell. Metastatic tumors were dramatically increased in metastasis model with DNP treatment, and phos-Ezrin Thr567expression in these metastatic tumor tissues increased. The results suggested that DNP could promote invasion and metastasis of nasopharyngeal carcinoma cells, and enhances phos-Ezrin Thr567expression, this implys that DNP-mediated NPC metastasis is associated with Ezrin Thr567phosphorylation.